Preparation of erythrocytes for immunochemical determination of human chorionic gonadotropin



United States Patent Ofifice 3,548,051 Patented Dec. 15, 1970 3,548,051 PREPARATION OF ERYTHROCYTES FOR IMMU- NOCHEMICAL DETERMINATION OF HUMAN CHORIONIC GONADOTROPIN David Dingwall, Hamilton, Scotland, assignor to Organon Inc., West Orange, N.J., a corporation of New Jersey No Drawing. Filed Mar. 17, 1967, Ser. No. 623,825 Claims priority, application Netherlands, Mar. 24, 1966, 6603909 Int. Cl. G01n l/00, 33/16 U.S. Cl. 4243 3 Claims ABSTRACT OF THE DISCLOSURE BACKGROUND OF THE INVENTION It is known that erythrocytes from mammals, such as cattle, horses, sheep, rabbits, as well as chickens and from human beings, can be used as an auxiliary in immunochemical determinations based on the reaction of an antigen with its antibody. The erythrocytes are applied, for instance, as carrier of antigens, for instance, human chorionic gonadotropin (HCG), serum gonadotropin, growth hormone, and further serum albumin, gamma globulins, insulin, luteinizing hormone and tetanus toxoid.

The use of the following tanning agents or mordants to improve the adsorptive properties of the erythrocytes is described in the literature: inulin, tannin, hydroquinone, bis-diazobenzidine, Formalin, acetaldehyde, pyruvic acid aldehyde and glyoxal. Also successive treatment of the erythrocytes with different tanning agents and mordants has already been applied, for example, a pre-treatment with Formalin, followed by reaction with tannin.

The last few years various publications have appeared about pregnancy tests to be performed in vitro, in which use is made of erythrocytes pretreated with a tanning agent or mordant and sensitised with HCG. The reaction thereof with homologous antiserum can be prevented by the HCG present in the urine to be examined, causing the formation of a specific sedimentation pattern of the erythrocytes on the bottom of the reaction vessel.

So far the erythrocytes sensitised after the pre-treatment have been stored as an aqueous suspension, but preferably in a freeze-dried form. But erythrocytes are very unstable particles and the preservation of these carriers has already required much study. The erythrocytes pretreated with a mordant are less unstable and can be readily freeze-dried, while in a freeze-dried state they are stable for a pretty long time, There exists a need of a presentation form, however, in which the erythrocytes can be well handled and are at the same time stable and have kept all their properties essential for the performance of immunochemical determination methods.

SUMMARY OF THE INVENTION Surprisingly a method has now been found for the preparation of erythrocytes suitable for immunochemical determinations of antigens by treating erythrocytes with one or more tanning agents, followed by sensitisation with the antigen, characterized in that the last stage of the sensitisation is carried out in the presence of formaldehyde in a concentration below 2% (weight/volume) at a pH between 5.5 and 8. Of the thus treated erythrocytes the mechanical properties have improved to such a degree that they can be pressed to tablets, the erythrocytes having kept their usability as an auxiliary in immunochemical reactions after suspension of these tablets.

It was already known from US. patent specification No. 3,096,250 to perform the sensitisation of erythrocytes pre-treated with Formalin in the presence of a Formalin solution, and that at a pH within .2 units of the isoelectric point of the antigen used. This means that when the antigen HCG with an isoelectric point of 2.98 is applied, sensitisation must take place below pH 5, unlike the present method, in which good results are only obtained at a pH over 5.5. Yet another difference is that according to the known method a higher Formalin concentration, viz. of 28%, is applied, While the new method utilises a concentration below 2%, and preferably below 0.25% (weight/volume). Finally the presence of formaldehyde is required throughout the sensitisation period according to the said patent, unlike the present invention.

Comparative tests have been performed with the present method of treatment, with HCG as antigen, at different pHs, the conditions being otherwise quite equal to those described in Example 1 of the said patent specification, from which it has clearly appeared that treatment at pH 4.4 and pH 5.0 does not lead] to erythrocytes usable for immunochemical determinations.

Further it has been found that excellent results are obtained with erythrocytes pre-treated successively with Formalin and tannin. This method lends itself particularly well for the use of human chorionic gonadotrophin (HCG) as antigen. An HCG concentration of 10-50 I.U./ml. is preferred. When very pure HCG is utilised the concentration must be raised to about 200 I.U./ml. During the sensitisation with HCG, which process usually takes one or a few days, the formaldehyde is allowed to act upon the erythrocytes during the last 12-24 hours or there abouts, but preferably during the last 15 hours. The temperature at which the action is optimal lies between 30 and C., but preferably a temperature of 37 is applied.

Further it has been found that when HCG is used as antigen, the treatment according to the invention always yields very good results at a pH of about 6.4.

Finally a process has been found for the manufacture of tablets or other solid dosage unit forms of erythrocytes suitable for immunochemical determinations, characterized in that erythrocytes prepared according to the invention are mixed with the auxiliaries required for tabletting and the immunochemical determination, and next compressed to dosage unit forms.

Numerous tests were carried out with suspensions of erythrocytes obtained after sensitisation, mostly after one or two weeks storage, further with. the powders obtained after freeze-drying of the suspension, also after one or two weeks storage, for instance and finally with the tablets obtained from these powders, weighing about 15 mg., in order to establish the optimal conditions of the present treatment. These tests were carried out as follows.

Of a suitable antiserum a series of different dilutions was made, to which a certain quantity of a urine without HCG was added. The preparation of erythrocytes to be tested was mixed with this mixture. Next it was examined whether the erythrocytes agglutinated completely at a high dilution of the antiserum. The same preparation of erythrocytes was also added to mixtures of the same dilutions of the antiserum and urine with HCG. The result was only considered acceptable if in a short time. for instance in less than 2 hours, the presence of 1 I.U. HCG per ml. could be shown, i.e, when the agglutination of the erythrocytes loaded with HCG with antiserum in a suitable dilution was completely inhibited with urine containing 1 I.U./ml.

Erythrocytes treated according to the invention have become strong enough to be compressed to, say, tablets. For this purpose the conventional auxiliaries can be applied, which must not be aggressive against erythrocytes and the antigen attached to them. It stands to reason that the bond between the antigen and the erythrocytes must not be affected adversely by the auxiliaries used and that they must not have a perceptible influence on the determinations for which the erythrocytes are intended, so that the sensibility, the accuracy and the velocity of the determination reaction is not essentially changed.

Further it has been found that erythrocytes treated according to the invention do not only have greatly improved mechanical properties, but also a greater sensibility to agglutination with antiserum. Thus complete agglutination could even have been obtained with a nontreated suspension of erythrocytes sensitised with HCG with a dilution of antiserum of while after treatment in accordance with the invention the same suspension caused the same reaction with a dilution of antiserum of from /7500 to A5000- The invention is not limited to application in the sensitisation of erythrocytes with HCG; it can also be applied in the sensitisation with any antigen that can stand up to the present treatment with formaldehyde.

For immunochemical determinations both the powder of sensitised erythrocytes obtained, for example, after freeze-drying or spray'drying of the suspension, can be applied and the tablets compressed therefrom.

As the powders obtained after freeze-drying of suspensions of erythrocytes are very hygroscopic, they are mixed and compressed with the other auxiliaries, preferably in an atmosphere with a slight relative degree of moisture, preferably to about It is advantageous to choose such ingredients and apply such conditions in compressing that the disintegration time of the tablets is short, for example, less than 3 minutes.

As examples of auxiliaries to be applied the following are mentioned, which are preferably applied in the preparation of tablets for the performance of pregnancy reactions. As fillers are amongst others usable; saccharose, manitol, lactose and ureum; as lubricants for example: boric acid, starch, Carboway 4000 (polyethylene glycol) and magnesium stearate, as disintegration agents for example: starch and alginic acid. Other ingredients are for instance substances used in the said determination, such as phosphates, for example, monopotassium phosphate and disodium phosphate, serving as buffers for the suspension prepared from the tablets, further sodium chloride and a chelating agent, such as the disodium salt of ethylene-diamine tetra acetic acid (E.D.T.A.) and albumin. The required ingredients can be mixed in a dry condition with the treated erythrocytes before tabletting, but it is more advantageous to dissolve the soluble ingredients together and to dry them by spray-drying, followed by mixing with the treated erythrocytes to obtain a very homogeneous mixture and to prevent the erythrocytes from being damaged during the further treatment by coarse crystalline components of the auxiliaries.

In the processing of insoluble auxiliaries special attention should be paid that they do not disturb the determinations performed with a suspension obtained from the tablets. For the insoluble components will be able easily to change the sedimentation pattern of the erythrocytes, thus influencing the accuracy of the observation unfavourably.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Further details of the process are to be found in the following examples which are to be regarded as illustrative and not as limiting.

EXAMPLE 1 A suspension of sheep erythrocytes treated in a known manner with Formalin and tannin was centrifuged and washed with a phosphate sodium chloride buffer of pH 6.4 and next incorporated in this buffer. To this mixture an equal volume was added of a solution of 50 I.U. HCG per ml. in a buffer of pH 6.4. This mixture was stored for 48 hours at 37 C., whereupon formaldehyde was added till a final concentration had been obtained of 0.25% (weight/volume) formaldehyde. Next this mixture was stored for 15 hours at a temperature of 37 C., whereupon the erythrocytes were again centrifuged, washed with a physiological salt solution, incorporated as 10% v./v. suspension in a physiological salt solution containing 0.1% of bovine serum albumin, and stored for a few days at 04 C.

After that the erythrocytes were centrifuged, washed with a physiological salt solution and freeze-dried in a known manner. The powder is a good reagent on HCG.

EXAMPLE II 300 ml. of the suspension of erythrocytes of pH 6.4 according to Example I were treated with 300 ml. of a solution of HCG with 50 I.U. HCG per ml. at pH 7.6 and next with formaldehyde in a final concentration of 0.5%. After freeze-drying the powder was tabletted. The tablets obtained gave excellent results as a reagent in immunochemical pregnancy tests.

EXAMPLE III By the method of Example I erythrocytes were sensitised with HCG in the presence of formaldehyde in a final concentration of 0.25%, and that for the last 24 hours at 30 C. After freeze-drying and compression tablets were obtained which gave excellent results as a reagent in an HCG test.

EXAMPLE IV Experiments were performed with treatment with formaldehyde for the last 15 hours of the sensitisation of erythrocytes with HCG in accordance with Example I, but this time at pH 5.5. The suspension was dried in a Niro spray-drier with an inlet temperature of C. and an outlet temperature of 75 C. The resulting powder was next mixed with 0.4% (weight-weight) potato starch and compressed to tablets of about 15 mg. They gave excellent results as a reagent in immunochemical pregnancy determinations.

What is claimed is:

1. In the process of preparation of erythrocytes sensitized with human chorionic gonadotropin and which are suitable for immunochemical determination of human chorinic gonadotropin, wherein said erythrocytes are pre liminarily treated with a tanning agent, and then sensitized with human chorionic gonadotropin, the improvement which comprises subjecting said erythrocytes to sensitization with human chorionic gonadotropin for a period of at least about 24 hours, and thereafter subjecting said erythrocytes for an additional 12 to 24 hours to the action of formaldehyde in a concentration between about 0.25% and about 2%, weight/volume, at a pH between about 5.5 and about 8, whereby said sensitized erythrocytes are rendered mechanically stable.

2. The process of claim 1 in which said sensitized erythrocytes are admixed with an auxiliary for tableting selected from the group consisting of saccharose, mannitol, magnesium stearate, and alginic acid, and then compressed into tablet form.

3. The solid tablet prepared by the process of claim 2.

Referenc es Cited Wide: Acta Endocrinologca, Supp. 70, vol. 41, 1962, UNITED STATES PATENTS 3,096,250 7/1963 Ingraham ALBERT T. MEYERs, Primary Examiner 3236732 2/1966 Arqmna 5 A. P. FAGELSON, Assistant Examiner OTHER REFERENCES us. c1. X.R. 77, 72 $1923; 1 2: bjkiififi? 1 94 3i 19 7 1 99? 16 Little: Tablet Making, The Northern Pub. Co. Ltd., Liverpool, England, pp. 48, 51, 62, 64 (1949). 

